The major hypothesis to be tested is that circulating prolactin (PRL) levels regulate the Hypothalamic- Pituitary-Adrenal (HPA) axis response to stress. Additionally, the mechanism of action of PRL on HPA axis activation occurs due to alterations in PRL transport into the brain via specific receptors in the choroid plexus. The duration of the PRL increase is important. The activation of the HPA axis in response to physical, systemic stress (restraint or shaker), will be determined in male and female mice that have different circulating PRL levels. Animals will be: (1) Normal prolactinemic: intact, PRL wild-type mice;(2) Hyperprolactinemic: (i) post- partum, female wild-type mice (physiologically hyperprolactinemic) or (ii) wild-type and PRL knockout mice treated with exogenous PRL to induce hyperprolactinemia for 1, 3 or 6 days to determine the time course and specificity of the inhibitory effects of hyperprolactinemia on the HPA axis;(3) Hypoprolactinemic: (i) PRL knockout mice or (ii) intact, PRL wild-type mice treated with PRL-Antiserum (PRL-AS) to abolish endogenous PRL levels. Although endogenous PRL will be depleted in this group, in contrast to PRL knockouts, they will have developed with normal PRL levels, and thus control for any compensatory mechanisms which may have developed in knockout mice. PRL-receptor (PRL-R) expression levels in the choroid plexus and CRH and PRL expression levels in the hypothalamic paraventricular nucleus (PVN) will be quantified using qRT-PCR to determine mRNA levels and western blot analysis or radioimmunoassay to determine protein levels. Plasma corticosterone (CORT) and PRL levels will also be measured. We predict that normal prolactinemic, wild type mice (controls) will show a normal stress response, but animals with sustained hyperprolactinemia will have an attenuated response to stress, possibly due to decreased PRL transport into the brain or decreased sensitivity of CRH neurons to stimulation. The response in the PRL knockouts is more difficult to predict because, to date, no studies have determined if PRL is essential for HPA axis activation. These proposed studies will address this question. To demonstrate the specificity of the PRL effect, endogenous PRL levels will be depleted in wild type control mice. Stress, a major disrupter of homeostasis is involved in a wide range of pathologies, and stress-related illnesses cost over $1 billion annually in the US. In spite of these devastating effects, many neuroendocrine mechanisms involved in regulating stress are not known. PRL, a well-characterized, reproductive hormone, is also an important stress hormone. While the critical roles the HPA axis plays in the stress response have been extensively studied, the role of PRL and especially the interactions between the HPA axis and PRL are poorly understood. PUBLIC HEALTH RELEVANCE Stress has a major, negative impact on society, costing over 1 billion dollars in health care costs annually in the US alone. Stress produces major deleterious effects and leads to many pathological conditions, including cardiovascular disease, impairment of the immune and reproductive systems, as well as sleep disorders, depression and substance abuse. The focus of these studies is on the neuroendocrine response to stress, particularly the intriguing connection between the "lactation hormone" prolactin and the primary stress pathways in the brain involving the control of corticosteroids. Although prolactin is an important stress hormone, its mechanisms of action and its interaction with the other stress hormones are poorly understood. Discovering the neuroendocrine mechanisms regulating the stress response will lead to improved treatments of stress-related disorders, and positively impact human health.